Journal of Research in Biology

ISSN No: Print: 2231 6280; Online: 2231- 6299

An International Scientific Research Journal

ORIGINAL RESEARCH

Journal of Research in Biology

Epidemiological studies on hospital based incidence of Japanese

encephalitis in the Jharkhand state of India
Authors:
Noman Alam Md1,
Sahu NP1 and
Sultan Ahmad Md2.

Institution:
1. Rajendra Institute of
Medical Science (RIMS),
Ranchi, Jharkhand, India
2. S N (PG) College,
Azamgarh, Uttar Pradesh,
India

Corresponding author:
Sultan Ahmad Md.

Email Id:

ABSTRACT:
Study was conducted in Rajendra Institute of Medical Science (RIMS), Ranchi,
Jharkhand, during June 2012 to September 2013. The objective of the study was to
know the hospital based incidence of Japanese Encephalitis (JE) and to study the age,
sex and seasonal pattern of infection. 219 cases were analyzed by the Department of
Microbiology, RIMS, Ranchi with clinical diagnosis. These samples were experimentally
tested to confirm Japanese encephalitis by IgM Antibody Capture Enzyme Linked
Immunosorbent Assay (MAC ELISA). Out of 219 cases, diagnosis was confirmed in 53
cases (24.20%) with male to female ratio of 0.89:1. All were below 15 yrs of age. Most
of the cases were children. Clinically, fever (100%), altered sensorium (69.80%)
headache (54.71%), neck rigidity (39.62%), Kernigs sign (28.30%), convulsion (43.39%)
and vomiting (35.80%) were the major findings observed. Majority of cases were from
rural areas. The hospital based incidence of JE was found to be significant in the area
of study. Effective measures should be taken to minimize disease transmission.

Keywords:
Japanese Encephalitis, hospital based incidence, Sensorium, Jharkhand,
Kernigs sign, Epidemic, seasonal pattern, pediatrics

Article Citation:
Noman Alam Md, Sahu NP and Sultan Ahmad Md.
Epidemiological studies on hospital based incidence of Japanese Encephalitis in the
Jharkhand state of India.
Journal of Research in Biology (2015) 5(1): 1611-1618
Dates:
Received: 05 Aug 2014

Web Address:
http://jresearchbiology.com/
documents/RA0469.pdf
Journal of Research in Biology
An International
Scientific Research Journal

Accepted: 30 Aug 2014

Published: 28 Jan 2015

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/

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Alam et al., 2015

in the transmission cycle with respect to human infection

INTRODUCTION

An important cause of admission, mortality and since they live in vicinity of humans, whereas herons or
permanent neurological sequel in the hospital is viral ardeid birds are important reservoirs. Horses are the only
encephalitis. Hundreds of known arthropod borne viruses other vertebrate that also develop Central Nervous System
(Arbo viruses) causes human diseases and among them (CNS) infection and are considered as dead end hosts,
the commonest virus is Japanese Encephalitis Virus (JEV) though the amphibians, reptiles and bats can also be
that causes childhood viral encephalitis worldwide.
In

India,

annual

incidence

of

infected by JEV. It does not cause encephalitis in pigs

Japanese and birds and rodents are relatively refractory to infection

encephalitis ranged between 1714 and 6594 (NVBDCP). (MacKenzie, 2005).

Epidemics are reported from many parts of Assam, Bihar,
Haryana, Tamil Nadu, Uttar Pradesh, West Bengal, MATERIALS AND METHODS
Manipur, Andhra Pradesh, Karnataka, Madhya Pradesh,
Orissa and union territory of Goa and Pondicherry

1. Place
The present study has been carried out in the

(Kabilan et al., 2004) and among them Uttar Pradesh and Department of Microbiology, Rajendra Institute of
Assam is the major affected state. In Jharkhand, first case Medical Sciences (RIMS), Ranchi, during the period of
was reported in 2010.

MD course. A total of 219 cases were included in this

Japanese encephalitis virus, a small enveloped study and the studied cases belong to patient reach in the
plus stranded RNA virus is an arthropod borne Arbo RIMS, Ranchi and sample reach from the different
virus, belonging to the family Flaviviridae and genus districts of Jharkhand. Selection of cases was done on the
Flavivirus. Japanese Encephalitis Virus (JEV) causes basis of clinical feature of encephalitis attending in the
inflammation of the brain, which may lead to permanent RIMS, Ranchi.
brain damage, and has a high mortality rate. In India, it 2. Selection of persons
was first recognized in 1955 when virus was isolated from There are certain criteria followed while selecting a
Culex vishnui from Vellore during the outbreak of person to include in the study. They were
encephalitis in Tamil Nadu (Namachivayam and Umayal, a. The person must be with the symptoms of encephalitis.
1982).

b. The person should have fever, headache, coryza or flue

Billions of people worldwide are living in JE like illness during febrile and acute presentation of

endemic area and it was estimated that JEV is responsible symptom of encephalitis like headache, nausea, diarrhea,
to cause 45000 cases of disease and 10,000 deaths per vomiting, myalgia, altered behavior, convulsions, coma
year (Solomon and Winter, 2004; Van den Hurk et al., and other neurological problems like ocular palsies,
2009). The majority of cases (about 85%) occur among hemiplegia, quadriplegia, dystonia, choreoathetosis and
children who were less than 15 years of age.
The transmission of JEV occurs through a

coarse tremors, etc.

Keeping the above points in mind, sample was

zoonotic cycle where mosquito acts as an intermediate collected

and

transported

to

the

Department

of

and replicative vector, whereas pigs and ardeid birds were Microbiology, RIMS, Ranchi, for the IgM detection
amplifying hosts. Humans are infected due to biting of an through ELISA against Japanese encephalitis. The
infected mosquito and are dead end hosts. There is no collection, transport and storage of specimens were done
proof of human to human transmission, possibly due to according to the standard procedures followed at National
the presence of transient viraemia. Pigs play a major role Institute of Virology (NIV), Pune (WHO, 1980).
1612

Journal of Research in Biology (2015) 5(1): 1611-1618

Alam et al., 2015

Blood specimen collection

al., 1988). It was observed that the cross reactive domain

1. Cerebrospinal fluid (CSF)

for Haemagglutination Inhibition A (HIA) was very

Cerebro Spinal Fluid (CSF) specimen was different for flavivirus. It was also observed that the JE
collected in sterile screw bottles under all aseptic virus specific domain for HIB was similar or continuous
precaution by trained persons. The containers were with the domains that was represented by non HI JE-virus
properly labeled and transported at earliest to the specific MAbs C and flavivirus cross-reactive MAbsD.
microbiology laboratory. All attempts were made to Domain E was expressed by two different MAbs that
collect CSF sample for the confirmation of diagnosis as reacted with both i,e, JE virus as well as uninfected cell
per the Guidelines for surveillance of acute encephalitis nucleus. On the basis of specificity, following conclusions
syndrome, government of India, 2006.

were made:

1.1Process of CSF Collection

1. Two different types of domain for antigen i.e. A and

Cerebrospinal fluid flows through subarachnoid B were associated with HI.

space and bathes and protects the brain and spinal cord. 2. A domain is also associated with ADPE.
Cerebrospinal fluid specimens were collected through 3. HI and NT were dissociated function in the in vitro and
lumber puncture by medical expert. The spinal needle is in vivo.
inserted between 3rd and 4th lumbar vertebrae under 4. All MAbs that react with epitopes in the B domain
aseptic condition. Once the needle was properly had HI and NT protective activity unable to show ADPE.
positioned in the subarachnoid space, pressure was

So, for the development of synthetic testing kit

measured and 2-3 ml of CSF is collected in the empty B is considered most suitable (Cecilia et al., 1988)
blood collecting vial and stored at +4C. Small amount of Negative controls
CSF was used for physical, cytological, biochemical, and

Heat inactivated serum. The JE detect negative

microscopic examination and the remaining CSF was control will aid in monitoring the integrity of the kit as
stored

aseptically

for

serology

and

viral

culture well. It was stored at 2-8C until ready to use for up to

examination.

7days. The vial was quick spin briefly before use to

1.2 Procedure for preparation of different compounds collect the content at the bottom.
for test

Positive controls
IgM Antibody Capture ELISA (MAC-ELISA)

Heat inactivated serum. The JE detect IgM

Kit was used supplied by InBios International Inc. (USA). positive control was added in monitoring the integrity of
Procedure was followed as recommended by InBios the kit as well. It was also stored at 2-8C until ready to
International Inc. (USA).

use for up to 7days. The vial was quick spin briefly before

Monoclonal Antibodies (MAbs) were raised use to collect the content at the bottom as recommended
against JE virus and were used to map topographically the by supplier.
epitopes on the envelope protein (Parida et al., 2005). Test methodology
Two separate clusters of epitopes were revealed. It was

Monoclonal antibodies (MAbs) were raised

observed that due to the Haemagglutination Inhibition against an Indian strain of JE virus and were used to map
(HI), Neutralization (NT) reactions that causes positive topographically the epitopes on the envelope protein
protection and Antibody Dependent Plaque Enhancement (Parida et al., 2005). Sample dilution buffer was used to
(ADPE) assays with the MAbs, there were five functional dilute specimen and antibodies. It is necessary to block
domains viz; A, B, C, D, and E were identified (Cecilia et unbound sites on the solid phase in order to minimize non
Journal of Research in Biology (2015) 5(1): 1611-1618

1613

Alam et al., 2015

-specific reaction. Serums were diluted at 1:100 and CSF amount of JE virus specific IgM antibodies in the sample.
at 1:10 with sample dilution buffer and the coated wells The positive and one negative control. were mainly for
were washed thrice with washing buffer. 50 l of diluted validation of kit.
samples was transferred to the appropriate wells and 50l Expected value are given below
was reconstituted and added in positive and negative Positive: OD value 0.5; Negative: OD value 0.18
control wells respectively. Test procedures were as Interpretation of the result
followed by standard methodology as shown below by
flow diagram

If OD value of the sample tested exceeds OD of

negative control by a factor 5 (Sample OD Negative OD

Anti-IgM Antibodies Bound To Solid Phase

5), the sample was considered as positive.

Limitations

Standards and Samples Added

Diagnosis of JE infection was not based on the

results of this test alone but in conjunction with

IgM* Anti-IgM Complexes Formed

physicians

clinical

impression.

Moreo ver,

epidemiological data and travel history to epidemic area

Unbound Sample Proteins Removed

was also considered before making the diagnosis. IgM

appears in circulations 3-5 days post onset. Therefore,

Anti-IgM-HRP Conjugate Added

date of collection of sample after onset of disease also

influences the interpretation of the results.

Anti-IgM-HRP* IgM* Anti-IgM Complexes Formed

Statistical Analysis
Students one tailed ttest was used and the level

Unbound Anti-IgM-HRP Removed

of significance was tested at <0.05 probability from

standard statistical tables (Fisher and Yates, 1963).

Chromogenic Substrate Added

RESULTS
Determine Bound Enzyme Activity

In this study, out of 219 cases tested from the

0.1 mg/ml TMB substrate was dissolved in 10 ml different districts of Jharkhand, Ranchi has maximum
citrate acetate buffer and further diluted with 25 l diluted number of 71suspected cases out of which, 16 cases were
(30%) H2O2 and 100 l TMB stock was prepared. TMB found positive for JEV. Latehar and Bokaro have 11.32%
was used for ELISA detection. Reaction between the positive cases each. Lohardaga, Palamu, and Ramgarh
substrate and immobilized Horse Radish Peroxidase have 5.67% positive cases each and Dumka, Koderma and
(HRP) conjugated secondary antibodies in the ELISA Saraikela have 1.88% positive cases each. No JE positive
wells produced a blue colored solution. After reaching the cases were found in Jamshedpur, Giridih, Godda, Jamtara,
desired color intensity, the reaction was stopped by adding Khunti, Pakur, Sahibganj, Simdega and Chaibasa (Table
acidic stop solution (1N H2SO4) which changed the 4) and for JE MAC ELISA, 53 were positive for JE
solution color from blue to yellow. The reactants were (24.20%) as shown in Table 1. Out of 109 CSF sample
allowed to remain stable for one hour and then the plate tested, 35 (66.03%) were positive for JE and out of 110
was analyzed on a microplate reader at 450 nm. Optical serum sample tested, 18 (33.96%) were positive for JE
densities were recorded as it was known that optical (Table 3). Age group distribution showed more number of
density of the samples is directly proportional to the cases between 3-8 yrs of age (66.02%) as shown in Figure
1614

Journal of Research in Biology (2015) 5(1): 1611-1618

Alam et al., 2015

Table 1. Incidence of Japanese Encephalitis during
June 2012 to September 2013 in the Jharkhand state
Total No. of
suspected cases
219

JE Positive cases

Percentage

53

24.20%

patients in the hospitals are found during monsoon months

(Figure 2).
Culex tritaeniorhynchus, C. gelidus and C.
pseudovishnui are found to be very efficient vectors for
JEV transmission (Solomon, 2004; 2006). Incubation

1. Out of 53 positive cases, percentage among male was period varies from 5-15 days. Due to sub clinical nature of
47.16% and among female was 52.83% i.e. the ratio was infection, symptomatic disease ranges from one out of
0.89:1(Table 2). Clinically, it was observed that all fifty to one out of thousands of human infections (Tsai,
patients with JE had a history of fever (100%), headache 2000). The onset of illness can be abrupt, acute, sub-acute
(54.71%), vomiting (35.8%), altered sensorium (69.8%), or gradual. Progression of disease can be divided into
convulsion (43.39%), neck rigidity (39.62) and kernigs three stages (i) Prodromal stage - characterized by high
sign

(28.30%)

(Table

5).

Our

findings

indicate, fever, headache, malaise, nausea and vomiting (ii)

statistically significant outbreak of JE in the survey area Encephalitic stage - diagnosed by altered sensorium, neck
as calculated t values were 6.839 in comparison to stiffness, tremor, muscular rigidity and speech impairment
tabulated value (2.069, df-23).

and (iii) Late stage - characterized by persistent sign of

Table 2. Gender wise distribution of cases during June CNS injury such as mental impairment, increased deep
2012 to September 2013 in the Jharkhand state
tendon reflexes, epilepsy and other behavioral
Sl.
No.

Gender

Total No.
of
suspected
cases
104

JE
Positive
cases

Percentage

1.

Male

25

47.16%

2.

Female

115

28

52.83%

Total

219

53

99.99%

abnormalities (Misra and Kalita, 2010). Case fatality rates

for JEV range from 0-30% (Burke et al., 1987). In a
study, chronic progressive encephalitis and relapses due to
perseverance of JEV in the CNS has been reported in a
few patients (Pradhan et al., 2001; Ravi et al., 1993) . Due
to shortcomes with other method, IgM ELISA test is the
only appropriate method for the provided samples that are
collected 3 to 5 days after infection. For example, after the

DISCUSSION

The monsoon season followed by an increase in first 9-10 days of illness, the presence of anti JEV IgM in
mosquito breeding due to water logging, leads to JEV CSF has sensitivity and specificity of >95% (Burke et al.,
spread in farm animals and results in human encephalitis 1985).
(Mani et al., 1991) in many parts of India as well as in

Out of 219 clinically suspected JE cases, our

other Asian countries (Erlanger et al., 2009; Hoke et al., study showed 24.20% serologically confirmed JE case
1988). Our observations also support this assumption and during June 2012 to September 2013 in Jharkhand. The
it was noticed that sudden increase in the number of disease was fond in children, as observed by earlier
Table 3. Ratio of different JE positive samples during
June 2012 to September 2013 in Jharkhand
Sl.
No
1.

2.

Specimen

worker elsewhere (Rashmi, 1999). This might be due to

high exposure to infected mosquito bites with lower
immunity. Female preponderance observed in this study

JE
Positive
18

Percentage

Serum

Total
specimen
110

33.96%

status which leads to higher exposure of female patient to

C.S.F.

109

35

66.03%

mosquito vectors.

Total

119

53

99.99%

Journal of Research in Biology (2015) 5(1): 1611-1618

might be due to poor caring, illiteracy, low socioeconomic

The clinical feature observed in this study was

1615

Alam et al., 2015

Figure 1 Age wise distribution of JE positive cases in the Jharkhand state of India
not uniform which might be due to the influence of host during monsoon has been shown to be followed by an
immune status, viral load and length of time between increase in mosquito, leading to sero conversion in farm
onset of disease and medical intervention. In our study, JE animals and later human encephalitis (Mani et al., 1991).
positivity was more in CSF when compared to serum
specimen which signifies the increase in number of cases CONCLUSION
of CNS infection with JE virus.

The hospital based incidence of JE was found to

Most of the positive cases have occurred from be significant in the area of study at 5% probability. JE is
July to September as shown in Figure 2, which commonest form of periodic and pandemic encephalitis in
corresponds to the monsoon or post monsoon season. It the south Asian region due to breeding of mosquito and
was reported in a previous study that the increased rainfall should be checked before considering the other viral

Figure 2 Month wise distribution of JE positive cases in Jharkhand, India

1616

Journal of Research in Biology (2015) 5(1): 1611-1618

Alam et al., 2015

Table 4. District wise distribution of JV positive cases in Jharkhand.
S.N Districts
Total
JV
Percent
suspected Positive
cases
cases
1.
Bokaro
10
06
11.32
2.
Chatra
05
02
3.77
3.
Deoghar
02
00
00
4.
Dumka
02
01
1.88
5.
Dhanbad
05
02
3.77
6.
Jamshedpur
09
00
00
7.
Giridih
08
00
00
8.
Gumla
06
01
1.88
9.
Garhwa
16
02
3.77
10. Godda
00
00
00
11. Hazaribagh
26
06
11.32
12. Jamtara
01
00
00
13. Kodarma
01
01
1.88
14. Khunti
01
00
00
15. Lohardaga
07
03
5.67
16. Latehar
17
06
11.32
17. Palamu
18
03
5.67
18. Pakur
00
00
00
19. Ranchi
71
16
30.19
20. Ramgarh
08
03
5.67
21. Sahebganj
00
00
00
22. Simdega
01
00
00
23. Saraikela
05
01
1.88
24. Chaibasa
00
00
00
Total
219
53
99.99
infections, and effective measure should be taken to
minimize disease transmission.
Table 5. Analysis of Various symptoms in
JE positive cases.
Sl. No.

Clinical feature

suffered a lot in the absence of any financial support.

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Journal of Research in Biology (2015) 5(1): 1611-1618